The long-term aim of this research program is to develop a quantitative understanding of eukaryotic gene regulation. In eukaryotes, the genomic DNA is wrapped in nucleosomes, which occlude much of the surface of the wrapped DNA and strongly distort it; yet, nucleosomes also recruit other proteins through interactions with their histone tail domains. Thus, the detailed locations of nucleosomes have important inhibitory and facilitatory roles in gene regulation, and is essential for the health and development of all eukaryotes. In the current project period we advanced the understanding of where nucleosomes are located along the DNA, the principles that govern these locations, and how these influence competition with other factors and gene expression. In the next grant period, we propose to extend these findings in three directions. Studies in Aim 1 will measure nucleosome-factor competition directly and quantitatively, genome-wide, as synchronized pools of cells progress through the cell cycle; and also at specific engineered transgenes, where we will measure not just nucleosome positioning and occupancy but also the identities, locations, and occupancies of bound factors. These data will test our current understanding of nucleosome-factor competition and the thermodynamic equilibrium hypothesis on which our ideas are based. Studies in Aim 2 seek to measure the exact locations of nucleosomes to the basepair. This information is needed in order to: understand where nucleosomes will or will not compete with factors binding at adjacent target sites; to define the intrinsic 3- dimensional structure of the chromatin fiber; to establish the detailed structural biology of promoters; and to define the true nucleosome-DNA sequence preferences. Studies in Aim 3 will develop and experimentally test a quantitative predictive model for predicting the gene expression output given the genomic DNA sequence, and the concentrations, binding sites, and affinities of a set of transcription factors active in the cell. PUBLIC HEALTH RELEVANCE: The proper regulation of genes is essential for the development and health of all organisms. We discovered a previously unrecognized aspect of gene regulation, in which the genomic DNA sequence influences its own organization into chromosomes, and thereby influences diverse essential chromosome functions. Our hope is that a better understanding of the nature of this novel information, and how it influences gene expression and DNA replication will lead, over the long term, to a better understanding of fundamental mechanisms in disease processes, and from there, to new diagnostics and therapeutics.